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Restriction Endonucleases >
Restriction Endonucleases >
BsgI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
Bacillus sphaericus B922 (H. Kong)
Reagents Supplied:
NEBuffer 4 (10X)
S-adenosylmethionine (SAM) (400 X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 50% | | NEBuffer 2: | | 75% | | NEBuffer 3: | | 50% | | NEBuffer 4: | | 100% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
| dam methylation: Not sensitive | | dcm methylation: Not sensitive | | CpG methylation: Not sensitive |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.50 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Supplemented with 80 μM S-adenosylmethionine (SAM)
Incubate at
37°C.
1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total volume of 50 µl.
Concentration:
3,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
200 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
0.05% Triton X-100
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent B
Notes
General notes:- Storage of SAM: S-adenosylmethionine is stored at –20°C as 32 mM solution dissolved in sulfuric acid (0.005 M) and 10% ethanol. SAM in this solution stored under ideal conditions remains active for up to 6 months. SAM is unstable at (pH 7.5), 37°C, and should be replenished for reactions incubated longer than 4 hours.
Many problems in achieving complete digestion can be alleviated by using fresh SAM.
- BsgI requires 80 µM S-adenosyl-methionine in reaction mixture for optimal activity (supplied with enzyme). Incubation without S-adenosylmethionine results in 25% activity.
- SAM should be kept frozen at shipping concentration and diluted prior to each reaction.
FAQs
- Why doesn't BsgI cut well?
- Are there known sequence errors at the recognition site in a database?
- What is the activity of BsgI at 25°C?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 3-fold overdigestion with BsgI, > 95%
of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM)
at 16ºC. Of these ligated fragments, approximately 75% can be recut with BsgI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 50 units of BsgI incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of BsgI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (105 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Reagents Sold Separately
NEBuffer 4
S-adenosylmethionine (SAM)
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